kato-hayashi_s. japonicum cell-free circulating antigen

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Acta Tropica 141 (2015) 178183

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Acta Tropica

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Detection of active schistosome infection by cell-free circulating DNAof Schistosoma japonicum in highly endemic areas in SorsogonProvince, the Philippines

Naoko Kato-Hayashi a , Lydia R. Leonardo b , Napoleon L. Arevalo c , Ma. Nerissa B. Tagum d , James Apin e , Lea M. Agsolid f , James C. Chua b , Elena A. Villacorte b , Masashi Kirinoki a ,Mihoko Kikuchi g , Hiroshi Ohmae h , Kosuke Haruki i , Yuichi Chigusa a ,a Laboratory of Tropical Medicine and Parasitology, Dokkyo Medical University, Mibu 321-0293, Tochigi, Japanb Department of Parasitology, College of Public Health, University of the Philippines Manila, 625 Pedro Gil St., Ermita, Manila 1000, the Philippinesc Center for Health Development No. 5, Department of Health, Sorsogon City, Sorsogon, the Philippinesd Municipal Health Ofce, Irosin, Sorsogon, the Philippinese Municipal Health Ofce, Juban, Sorsogon, the Philippinesf Provincial Health Ofce, Sorsogon City, Sorsogon, the Philippinesg Department of Immunogenetics, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Sakamoto, Nagasaki 852-8523, Japanh Department of Parasitology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japani Department of Clinical Laboratory, Dokkyo Medical University Koshigaya Hospital, Koshigaya 343-8555, Saitama, Japan

a r t i c l e i n f o

Article history:Available online 15 May 2014

Keywords:Schistosomiasis japonicaDiagnosisActive infectionCell-free circulating parasite DNASerumUrine

a b s t r a c t

The current status of schistosomiasis in highly endemic areas is difcult to determine by ovum detec-tion because of the supercially low parasite load after mass drug administration, whereas the parasitetransmission rates are still high. Cell-free parasite DNA is fragments of parasite-derived DNA existing inthe hosts body uids. We conducted population-based studies to test the presence of cell-free schisto-some DNA in endemic areas of Sorsogon Province, the Philippines. Schistosome DNA in the serum and

urine of KatoKatz (KK)-positive subjects was detected by PCR (100% sensitivity). Schistosome DNA wasalso detected from KK-negative subjects (9/22 serum and 10/41 urine samples). Schistosome DNA wasfound to be network echogenicpattern (NW)-positive (serum53.3%, urine 42.9%)or NW-negative (serum25.5%, urine 20.8%) and enzyme-linked immunosorbent assay (ELISA)-positive (serum 47.1%, urine 40%)or ELISA-negative (serum 33.3%, urine 13.3%). These results indicate that cell-free schistosome DNA is apromising diagnostic marker for active schistosome infection in the case of light infection.

2014 Elsevier B.V. All rights reserved.

1. Introduction

Schistosomiasis is a major parasitic disease affecting approxi-mately 240 million people worldwide, with more than 700 millionpeopleatriskofinfection( WHO Schistosomiasis,2011 ). Thediseaseresults indiversehealth andsocioeconomicproblems,ranging fromatelioses to the death of the infected individuals and workforcereduction. In recent years, the disease has also become a health

Some parts of this study were presented at the Report on the CollaborativeProject, Institution of Tropical Medicine (Nekken), Nagasaki University, Japan(2010), the Proceedings of the Annual Meeting of Japanese Society of Parasitology(2011, 2013, Tokyo, Japan) and the RNAS+ Meeting (Hanoi, 2012).

Corresponding author. Tel.: +81 282 87 2134; fax: +81 282 86 6431.E-mail address: [email protected] (Y. Chigusa).

threat in non-endemic areas as a result of parasite importation(Clerinx and Gompel, 2011; Wichmann et al., 2013 ).

Most of the current diagnostic methods are concerned withdetection of the presence of schistosome ova or parasite-specicantibodies by microscopy or immunological methods, respectively(Doenhoff et al., 2004 ). Pathological lesions are diagnosed as char-acteristic images by ultrasound (US) or computed tomography andconrmed by biopsy ( Fig. 1). Although the current gold standardfor diagnosing schistosomiasis is ovum detection, it is not applica-ble in the early stage of infection because of the absence of parasiteovathat cause thepathologicallesions.Furthermore,the symptomsandsigns inthe early stageof infectionmimic those of various otherdiseases. Therefore,it is important to develop earlydiagnostic toolsthat are not dependent on the presence of ova.

Cell-free circulating nucleic acids are fragments of nucleic acidsthat have been liberated from cells and exist in the bloodstream,

http://dx.doi.org/10.1016/j.actatropica.2014.05.0030001-706X/ 2014 Elsevier B.V. All rights reserved.
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Fig.1. Schematicdiagram of diagnosticexaminationsand treatmentof schistosomiasis. Current diagnosticsfor schistosomiasisare detectionof parasite ova,parasite-specicantibodies and pathological lesions. All three diagnostics involve ova-related phenomena, and their results do not always reect active schistosome infection.

urine andotherbody uids. Recent studies havedemonstrated thatcell-free circulating nucleic acids in plasma/serum and urine areuseful as molecular diagnostic tools in oncology, prenatal diagno-sis, transplantation and other clinical areas ( Botezatu et al., 2000;Chan et al., 2003 ). Furthermore, parasite-derived cell-free circulat-ingDNA canbe detected in patient plasma, urine and saliva( Ponteset al., 2002; Gal and Wainscoat, 2006; Mharakurwa et al., 2006;Nwakanma et al., 2009; Buppan et al., 2010; Parija and Khairrnar,2007; Khairnarand Parija, 2008; Sandoval et al., 2006a; Wichmannet al., 2009, 2013; Enk et al., 2012; Lodh et al., 2013; Kato-Hayashiet al., 2013 ). We have demonstrated schistosome DNA in theserumand urine of infected animals 1 day after infection ( Kato-Hayashiet al., 2010 ). These ndings suggest that cell-free circulating schis-tosome DNA in host body uids may indicate active schistosomeinfection. We conducted preliminary population-based studies todetect active schistosome infection using cell-free circulating DNAof Schistosomajaponicum inhighly endemic areas of thePhilippines.

2. Materials and methods

2.1. Study areas

The studies were conducted in villages (barangays) endemicfor Schistosoma japonicum in Sorsogon Province, which is locatedon the southern Luzon Island of the Philippines. The barangayswere Bacolod, Bagsangan, Bolos, BuenaVista, Bura Buran, Carriedo,Gumapia, Guruyan, and San Pedro.

2.2. Examinations

Theparticipantswere examinedby abdominal US examinations,enzyme-linked immunosorbent assays (ELISAs) and KatoKatz(KK)stooltests( Endiss et al.,2005 ) usingsinglestool samples. In therst study in 2009 (Study 1), schistosome DNA detection was per-formed witha focus on those participantswho werediagnosedwithsevere hepatic brosis. Hepatic brosis is characteristic of chronicschistosomiasis and is triggeredby parasiteova.The hepatic lesionshave a network echogenic pattern (NW) in case of infection with

S. japonicum (US type 3) ( Ohmae et al., 1992 ). This can indicate

either a number of infections over a period of time or even long-standing infection. Twenty-three participants (21 males and twofemales, 1663yearsof age) were enrolled (23 serum and sixsalivasamples). In the second study in 2012 (Study 2), schistosome DNAdetection was performed with a focus on those who lived in theBagsangan barangay because, according to previous surveys, thisbarangay was highly endemic for schistosomiasis and because weexpected more patients here to test positive for schistosome ova.Forty-ve participants (29males and16 females, 674 years of age)were enrolled (45 urine samples). The participants were enrolledin only a single study and not both.

In parallel with the examinations, each participant was given aquestionnaire regarding the symptoms of their illness thatinferredschistosome infection, e.g., bloody stool, abdominal pain, hae-matemesis, dizziness, headache, convulsion, paralysis and speechdisturbance and the experience of praziquantel (PZQ) treatment.Furthermore, they were asked about the presence of snail coloniesin their daily living area and their experiences with farm work.

Before these examinations, informed consent was obtainedfrom all of the participants. This study was approved by theBioethics Committee of Dokkyo Medical University (approval No.1969).

2.3. ELISA

For convenience of transportation and storage, blood sam-ples for the ELISA test were obtained on a piece of lter paper(Advantec Blood Sampling Paper Type I, Toyo Roshi Kaisha, Ltd., Japan) and allowed to dry at room temperature (RT). A parasite-specic antibody in the serum was tested by standard ELISA(Matsuda et al., 1984 ). To prepare the samples for the assay, a3.0-mm-diameter disc of blood-soaked lter paper (approx. 4 lof whole blood, i.e., approx. 2 l of serum) was punched out andextracted in 600 l of extraction buffer (1% BSA, 0.05% Tween20, 0.5% skim milk in PBS) overnight at 4 C. Polystyrene 96-wellELISAplates (GreinerBio-One,Co.,Ltd., Germany)were coated with100 l/well of S. japonicum soluble egg antigens (SEA, 10 g/ml)in 0.05 M carbonate bicarbonate buffer (pH 9.6) and incubated at

37

C and then left overnight at 4

C. The plates were then washed


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Table 1Comparative evaluation of PCR and conventional examinations.

Study 1

KK PCR

Positive rate + Total Sensitivity(95% CI)

Specicity(95% CI)

PPV (95% CI) NPV (95% CI) PLR (95% CI) NLR (95% CI) + Total Sensitivity(95% CI)

Specicity(95% CI)

P

PCR (serum) 43.5% ( n = 23) + 1 9 10 100% 59.1% 10.0% 100% 2.4 0 0 13 13 (1. 3100) (35.479.3) (0.344.5) (66.1100) (1.44.0)

NW 65.2% (n = 23) + 1 14 15 100% 36.4% 6.7% 100% 1.6 0 8 7 15 80.0% 46.2% 53 0 8 8 (1.3100) (17.259.3) (0.231.9) (51.8100) (1.12.2) 2 6 8 (44.497.5) (19.274.9) (26.678.

ELISA 73.9% (n = 23) + 1 16 17 100% 27.3% 5.9% 100% 1.3 0 8 9 17 80.0% 30.8% 47 0 6 6 (1.3100) (10.750.2) (0.128.7) (42.1100) (1.11.8) 2 4 6 (44.497.5) (9.161.4) (2372.2)

KK 4.3% (n = 23) + 1 0 1 10.0% 100% 10 9 13 22 (0.344.5) (66.1100) (1.3

Study 2KK PCR

Positive rate + Total Sensitivity(95% CI)

Specicity(95% CI)

PPV (95% CI) NPV (95% CI) PLR (95% CI) NLR (95% CI) + Total Sensitivity(95% CI)

Specicity(95% CI)

P

PCR (urine) 31.1% ( n = 45) + 4 10 14 100% 75.6% 28.6% 100% 4.1 0 0 3 1 3 1 (28 .4 1 00 ) (59 .7 8 7.6) (8.4 58 .1 ) (83 .8 1 00 ) (2.4 7.0)

NW 46.7% (n = 45) + 2 19 21 50.0% 53.7% 9.5% 91.7% 1.1 0.9 9 12 21 64.3% 61.3% 42 2 2 2 24 (6.893.2) (37.469.3) (1.230.4) (7399) (0.43.0) (0.32.6) 5 19 2 4 (35.187.2) (42.278.2) (21.866.0

ELISA 66.7% (n = 45) + 3 27 30 75.0% 34.1% 10.0% 93.3% 1.1 0.7 12 18 30 85.7% 41.9% 40 1 1 4 1 5 (19 .4 9 9.4) (20 .1 5 0.6) (2.1 26 .5 ) (68 .1 9 9.8) (0.6 2.1) (0.1 4.2) 2 1 3 15 (57 .2 9 8.2) (24 .5 6 0.9) (22 .7 5 9.4

KK 8.9% (n = 45) + 4 0 4 28.6% 100% 1 10 31 41 (8.458.1) (83.8100) (28.4

KK: schistosome ova detected by KatoKatz stool test, PCR: cell-free circulating schistosome DNA detected by polymerase chain reaction, NW: ova-induced pathology detected by ultrasonographyELISA: schistosome-specic antibody detected by enzyme-linked immunosorbent assay PPV: positive predictive value, NPV: negative predictive value, PLR: positive likelihood ratio, NLR: negativinterval.


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three times with PBST (0.05% Tween 20 in PBS), blocked with120 l/well of 1% BSA in PBST and incubated for 15min at RT.After being washed three times, 100 l/well of samples was addedto the plates, which were then incubated for 45 min at 37 C.After being washed three times, horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Cappel, USA) was diluted 1:2000with 1% BSA in PBST, and 100 l was added to each well, and theplateswereincubated for1 h at37 C. Then,200 l/well of substratesolution containing 0.03% of 2,2 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS; Sigma-Aldrich Co., USA) and 0.003% H 2 O2in 0.1M citrate phosphate buffer (pH 5.0) wasadded and the platesincubated for 1 h at RT. Optical density (OD) was measured at415 nm. The mean OD value of the positive controls in each platewas adjusted to 1.064, and concomitantly, the rest of the originaldata were adjusted. OD values of 0.200 were considered positive.

2.4. DNA extraction and PCR amplication

Because it is difcult to detect schistosome DNA from a smallamount of blood on a piece of lter paper, blood was collectedusing a syringe, and the serum was separated by centrifugation.Urine (3.5 ml)was concentrated to 140 l using an Amicon Ultra-15 Centrifugal Filter Device, 100K (Merck Millipore Ltd., Ireland).The total DNA from serum (140 l) and urine was extracted byusing a QIAamp Viral RNA Mini Kit (QIAGEN Sciences, Maryland,USA). Approximately 2ml of the saliva sample was collected usingan Oragene DNA kit (DNA Genotek Inc., Canada) and stored at RT.DNA was extracted according to the manufacturers instructions.

The primer pair CF (5 -GATCGTAAATTTGGA/TACTGC-3) and CR (5 -CCAACCATAAACATATGATG-3) was designed to detect part of the schistosome mitochondrial cytochrome c oxidase subunit 1(CO1) gene, which is common in at least four human schistosomes(253bp: Schistosoma mansoni ; 254bp: Schistosoma haematobium ,Schistosoma japonicum , and Schistosoma mekongi ) (Kato-Hayashiet al., 2010 ). PCR was performed in a nal volume of 20 l con-taining 2 l of 10 PCR buffer, 2.5 mM MgCl 2 , 0.2 mM aliquots of each dNTP, 0.5U of Platinum Taq DNA polymerase (Invitrogen TM,USA), 0.5- M aliquots of each primerand 2 l of template DNA. Thereactions were performed initially at 94 C for 2 min, followed by50 cycles of 94 C for 30s, 58 C for 30s, 72 C for 60 s and a further72 C for 7 min. The PCR products were identied by electrophore-sis on 2%agarose in TAE gels with 0.3 g/ml ethidium bromide andthen visualised under UV light.

2.5. Statistical analyses

All statistical analyses were performed with EZR (SaitamaMedical Center, Jichi Medical University; http://www.jichi.ac.jp/saitamaHP.les/statmedEN.html ; Kanda, 2013 ), which is a mod-ied version of R commander (The R Foundation for StatisticalComputing) and designed for implementing statistical functions

frequently used in biostatistics.

3. Results

Study 1 focused on subjects who had severe hepatic brosis(NW-positiverate:65.2%,15/23).Only oneparticipant (4.3%) testedpositive for schistosome ova by the single KK test, whereas 17 par-ticipants (73.9%) tested positive by ELISA. Schistosome DNA wasdetected in serum samples of 10 of the 23 participants (43.5%)(Table 1 ). Furthermore, schistosome DNA was detected in 2 of the6 saliva samples. In Study 2, the positive rate of single KK, NW,ELISA and PCR was 8.9% (4/45), 46.7% (21/45), 66.7% (30/45) and31.1% (14/45), respectively ( Table 1 ). Table 2 shows a compari-son between schistosome DNA detection by the above methods.

Schistosome DNA in serum and urine of KK-positive participants

Table 2Comparison of diagnostic tests of Schistosoma japonicum infection.

Study 1 Study 2

KK NW ELISA PCR (serum) KK NW ELISA PCR (urine)

+ +

+ + + 1 0 + + + 1 0+ + 0 0 + + 1 0+ + 0 0 + + 2 0

+ 0 0 + 0 0 + + 6 7 + + 7 10 + 1 0 + 0 2 + 1 2 + 2 8 1 4 1 11

Total 10 13 Total 14 31

KK: schistosome ova detected by KatoKatz stool test, NW: ova-induced pathologydetected by ultrasonography as network echogenic pattern, ELISA: schistosome-specic antibody detected by enzyme-linked immunosorbent assay, PCR: cell-freecirculating schistosome DNA detected by polymerase chain reaction.

was detected by PCR (100% sensitivity); schistosome DNA was alsodetected in KK-negative participants (9 of 22 serum samples and10 of 41 urine samples). Schistosome DNA was found to be NW-positive (serum 53.3%, urine 42.9%), NW-negative (serum 25.5%,urine 20.8%), ELISA-positive (serum 47.1%, urine 40%) and ELISA-negative (serum 33.3%, urine 13.3%). Table 3 shows the number of participants who had predictable symptoms of schistosome infec-tion and experiences of previous PZQ treatment. The majority of participants worked on farms (95.7% and 86.7% in Study 1 andStudy 2, respectively) and reported encountering snail colonies intheir daily living areas (73.9% and 91.1% in Study 1 and Study 2,respectively).

4. Discussion

Currently, ova detection methods, such as the KK test, are stillthe gold standard for detection of active schistosome infection inconventional diagnostics. In the schistosomiasis-endemic areas of Southeast Asian countries, including the Philippines, mass drugadministration (MDA) with PZQ is the main component of the con-trol program ( Tallo et al., 2008; WHO Schistosomiasis, 2011 ). Morethan 80%of participantsin thepresent study hadpreviouslyunder-gone PZQtreatment ( Table 3 ). As conrmed in previous studies andour present study, stool examinations, especially for a single KKtest,may notdetectlight infections inpatientswho haveundergoneMDA (Lin et al., 2008; Sinuon et al., 2010; Han et al., 2012 ) and inthose with chronic infections. Overall, ova detectionis notuseful inthe earlystages of infection (prepatent period). AlthoughELISA andUS examination are useful methods for schistosomiasis detection,their results donot always reectactive infection( Fig.1). Advancedliver brosis detected by US is irreversible after PZQtreatment, andit takes several years for the positive ELISA results to turn nega-

tiveafterthe treatment. Patientswithchronicinfections, especiallythose in high transmission areas, may become re-infected aftertreatment. In these patients, the new active infection and efcacyof PZQ cannot be evaluated by US and ELISA.

Cell-free schistosome DNA originates from worm and ovametabolites, excrement and/or cell debris. Its presence has beenreported in patient serum and urine ( Pontes et al., 2002; Sandovalet al.,2006a;Wichmann et al.,2009; Lodhet al.,2013; Kato-Hayashiet al., 2013 ). The results from studies of experimentalanimals haveindicated thatcell-free schistosome DNA is detectable in prepatentperiods ( Sandoval et al., 2006b; Suzuki et al., 2006; Xia et al.,2009; Kato-Hayashi et al.,2010 ). Accordingly,we propose thatcell-free schistosome DNA is a feasible marker for detection of activeschistosome infection independent of parasite ova. Although the

concentration of schistosome DNA fragments in host body uids
http://www.jichi.ac.jp/saitamaHP.files/statmedEN.html;http://www.jichi.ac.jp/saitamaHP.files/statmedEN.html;http://www.jichi.ac.jp/saitamaHP.files/statmedEN.html;http://www.jichi.ac.jp/saitamaHP.files/statmedEN.html;


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Table 3Summary of questionnaire responses of participants regarding their experience of symptoms common in schistosome infection.

Present symptoms and signs (%) Experience of PZQ treatment(%)

Snail colony indaily livingarea (%)

Experience of farm work (%)

Bloody stool Abdominalpain

Haematemesis Dizziness Headache Convulsion Paralysis Speechdisturbance

Study 1 0 (0) 2 (8.7) 0 (0) 4 (17.4) 6 (26.1) 8 * (34.8) 2 * (8.7) 1 * (4.3) 22 (95.7) 17 (73.9) 22 (95.7)KK+ 0 0 0 0 0 0 0 0PCR+ 0 1 0 2 3 4 2 1

Study 2 2 (4.4) 6 (13.3) 2 (4.4) 15 (33.3) 14 (31.1) 2 (4.4) 2 (4.4) 1 (2.2) 36 (80.0) 41 (91.1) 39 (86.7)KK+ 1 0 0 1 1 0 0 0PCR+ 1 2 0 3 4 1 1 1

KK+: numberof theparticipantswithclinical ndings plus schistosomeova by KatoKatzstooltest; PCR+: numberof theparticipants with clinical ndings plus schistosomeDNA by polymerase chain reaction.

* Number of participants with previous and/or present clinical ndings.

may depend on parasite load, it is likely to be in minute amounts,as demonstrated by Wichmann et al. (2009) , with stable resultsfrom relatively large amounts of patient serum (20ml). Thus, theconcentration and purication of sample DNA is required, exceptfor cases of high parasite load, immediately after infection or withanthelmintic administration. In the present study, we detectedschistosome DNAfrom relativelysmall amountsof samples (serum,140 l; saliva, approx. 250 l; urine, 3.5ml).

Detection of parasite-derived DNA in urine and saliva wasrecently reported in several studies ( Mharakurwa et al., 2006;Nwakanma et al., 2009; Buppan et al., 2010; Parija and Khairrnar,2007; Khairnar and Parija, 2008; Sandoval et al., 2006a; Lodh et al.,2013; Kato-Hayashi et al., 2013 ) indicating the feasibility of apply-ing DNA detection methods to schistosomiasis. This method hasthe additional benet of non-invasive collection of urine and salivasamples, resulting in the increase in cooperation of patients, andneitherspecial equipmentnor special trainingare required forsam-ple collection. Moreover, using urine and salivasamples over bloodreduces the biohazard risk to examiners.

Field applications of molecular-based detection of schistoso-

miasis mainly involve the detection of parasite ova-derived DNAfrom stool samples ( Pontes et al., 2003; Gomes et al., 2009; Funget al., 2012; Carneiro et al., 2013 ). The most frequent use of cell-free schistosome DNA is for detection of imported schistosomiasisin non-endemic countries ( Sandoval et al., 2006a; Wichmannet al.,2009, 2013; Kato-Hayashi et al., 2013 ), and to date, very feweld studies have employed this method ( Lodh et al., 2013 ). Ourstudy aimed at detecting active infection by targeting cell-freeschistosome DNA in body uids (serum, urine and saliva) in S. japonicum -endemic areas. Our results indicate a higher detectionrate of active infections compared with the KK test (43.5% vs. 4.3%inStudy1; 31.1% vs. 8.9%in Study 2)( Table 1 ). The sensitivity of PCR was 100% for both Study 1 and 2, and the specicity was 59.1% inStudy 1 and 75.6% inStudy2, based ona singleKK test( Table 1 ). The

relatively low specicities can be attributed to the verication biasand low sensitivity of the KK test. Schistosome DNA was detectedfrom KK-negative subjects (nine of 22 serum and 10 of 41 urinesamples) ( Table 2 ). Some participants who reported neurologicalsymptoms, including convulsion, paralysis and speech disturbancehad negative results for the KK test; however, cell-free schisto-some DNA was detected in their serum/urine ( Table 3 ). Hrteret al. (2014) demonstrated diagnosis of neuroschistosomiasis byschistosome DNA in the cerebrospinal uid and serum. Cell-freeschistosome DNA is not only sensitive but may also helpful in dif-ferential diagnosis, especially for cerebral schistosomiasis. It is alsoavailable for therapy evaluationafter PZQtreatment( Kato-Hayashiet al., 2013 ).

In schistosomiasis-endemic areas, annual MDA is insufcient

for disease elimination as the inhabitants are constantly at risk of

infection or re-infection. Thus, a comprehensive approach includ-ing accurate diagnostics, control of snail and animal reservoirhosts, improvements in sanitation, and public health educationis required. Cell-free schistosome DNA is a promising diagnosticmarker for indication of active schistosome infection even in casesinwhich the parasiteova aredifcult todetect because of PZQinter-vention such as in MDA situations. The use of cell-free schistosomeDNA as a diagnostic marker is necessary to obtain better infor-mation regarding the status of schistosome infections in endemicareas. Further investigation of its application in endemic areas isrequired.

Acknowledgements

This work was supported by the Collaborative Project, theInstitute of Tropical Medicine (Nekken), Nagasaki University (22-ippan-25); Health Labour Sciences Research Grant, the Ministry of Health, Labour and Welfare, Japan (H20-Shinkosaiko-Ippan-016);and the Japan Society for the Promotion of Science (JSPS) KAKENHI(No. 24590510).

We would like to thank the staff of the Provincial Health Ofce,Sorsogon and the Municipal Health Ofce, Irosin and Juban, Sor-sogon, the Philippines for their great help. We also thank theresidents of the schistosomiasis-endemic barangays in SorsogonProvince, the Philippines. We also thank Dr. Ken-Ichi Manaka of the Research Support Center, Dokkyo Medical University for tech-nical support and our laboratory coworkers, Dr. Satoru Kawai, Ms.Mayumi Ohshita, and Ms. Mayu Tanaka, at the Laboratory of Tropi-cal Medicine and Parasitology, Dokkyo Medical University for theirassistance.

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